Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia

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Gene expression signatures define novel oncogenic pathways

in T cell acute lymphoblastic leukemia

Adolfo A. Ferrando,

1

Donna S. Neuberg,

2

Jane Staunton,

3

Mignon L. Loh,

4,8

Christine Huard,

3,9

Susana C. Raimondi,

5

Fred G. Behm,

5

Ching-Hon Pui,

6

James R. Downing,

5

D. Gary Gilliland,

4

Eric S. Lander,

3

Todd R. Golub,

1,3

and A. Thomas Look

1,7

1Department of Pediatric Oncology 2Department of Biostatistical Science

Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115

3Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research, Cambridge, Massachusetts 02142 4Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115

5Department of Pathology

6Department of Hematology/Oncology

St. Jude Children’s Research Hospital, Memphis, Tennessee 38105

7Correspondence: thomas_look@dfci.harvard.edu

8Present address: Department of Pediatrics, University of California, San Francisco, California 94143

9Present address: Millennium Pharmaceuticals, Inc., Predictive Medicine Group, Cambridge, Massachusetts 02139

Summary

Human T cell leukemias can arise from oncogenes activated by specific chromosomal translocations involving the T cell

receptor genes. Here we show that five different T cell oncogenes (HOX11, TAL1, LYL1, LMO1, and LMO2) are often

aberrantly expressed in the absence of chromosomal abnormalities. Using oligonucleotide microarrays, we identified

several gene expression signatures that were indicative of leukemic arrest at specific stages of normal thymocyte

develop-ment: LYL1

signature (pro-T), HOX11

(early cortical thymocyte), and TAL1

(late cortical thymocyte). Hierarchical clustering

analysis of gene expression signatures grouped samples according to their shared oncogenic pathways and identified

HOX11L2 activation as a novel event in T cell leukemogenesis. These findings have clinical importance, since HOX11

activation is significantly associated with a favorable prognosis, while expression of TAL1, LYL1, or, surprisingly, HOX11L2

confers a much worse response to treatment. Our results illustrate the power of gene expression profiles to elucidate

transformation pathways relevant to human leukemia.

Introduction

2000; Rivera et al., 1991). Securing further advances in treatment

outcome will likely prove difficult without improved knowledge

of the factors that contribute to the malignant behavior of

trans-T cell acute lymphoblastic leukemia (trans-T-ALL) is a malignant

dis-formed thymocytes. Unfortunately, most of the clinical and

labo-ease of thymocytes, accounting for 10%–15% of pediatric and

ratory features that guide therapy for B cell precursor ALL are

25% of adult ALL cases. Patients with T-ALL tend to present

only marginally useful in T-ALL (Pullen et al., 1999).

with very high circulating blast cell counts, mediastinal masses,

Current understanding of the molecular basis of T-ALL has

and central nervous system involvement. The prognosis of

come largely from analysis of recurrent chromosomal

transloca-T-ALL in children and adolescents has improved in recent years

tions and intrachromosomal rearrangements. These

abnormali-due to intensified therapies, with 5 year relapse-free survival

ties typically juxtapose strong promoter and enhancer elements

rates now in the range of 60%–75% (Pui and Evans, 1998;

Silverman et al., 2001; Chessells et al., 1995; Schrappe et al.,

responsible for high levels of expression of T cell receptor genes

S I G N I F I C A N C E

Careful analysis of clonal chromosomal abnormalities in leukemic blast cells has been a catalyst for the development of new diagnostic and therapeutic strategies. However, this line of research has had a much greater impact on the B lineage leukemias than on T cell acute lymphoblastic leukemia (T-ALL), whose pathogenesis and molecular subtypes remain largely undefined. Using a combination of DNA microarray and RT-PCR methods to analyze clinical T-ALL samples, we obtained results in support of our central hypothesis that aberrant activation of certain key transcription factor genes, often in the absence of chromosomal rearrangements, is the principal transforming event in this disease. These developmentally important molecules are shown to drive a limited number of oncogenic pathways with prognostic significance. The ability to classify T-ALL according to shared pathways of leukemic transforma-tion has important implicatransforma-tions for future research. It provides a conceptual framework in which to identify specific genes that determine treatment responsiveness and should foster the development of successful new therapies directed to critical molecules in pathologic transcriptional cascades. The hypothesis-driven approach to microarray analysis described in this report may also be useful for the study of other types of human cancers.

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next to a small number of developmentally important transcrip-

mosome band 1p32 that are known to cause aberrant

expres-sion of TAL1 in T-ALL. However, 9 (31%) of the 29 cases with

tion factor genes, including HOX11/TLX1, TAL1/SCL, TAL2,

LYL1, BHLHB1, LMO1, and LMO2 (Finger et al., 1989; Xia et

increased expression of TAL1 (Figure 1B) had the Tal1d variant,

which results from a small deletion next to the TAL1 locus (Aplan

al., 1991; Mellentin et al., 1989; Wang et al., 2000; McGuire et

al., 1989; Royer-Pokora et al., 1991; Dube et al., 1991; Hatano

et al., 1990; Bernard et al., 1991). Thus, the majority of cases

with high levels of TAL1 oncogene expression (22/29) lacked

et al., 1991; Fitzgerald et al., 1991; Kennedy et al., 1991; Lu et

al., 1991; Brown et al., 1990; Aplan et al., 1991, 1992; Baer,

cytogenetic or molecular evidence of rearrangements affecting

the TAL1 locus, in agreement with results of an earlier study

1993; Begley et al., 1989; Chen et al., 1990; Greenberg et al.,

1990; Boehm et al., 1991), resulting in their aberrant expression

(Bash et al., 1995).

Thirteen (22%) of the 59 cases were classified as LYL1

in developing thymocytes. Although the oncogenicity of these

proteins is well established (Larson et al., 1994; Neale et al.,

(Figure 1C) on the basis of LYL1 expression levels that exceeded

the mean value in normal thymocytes by more than 5-fold.

1995; McGuire et al., 1992; Chervinsky et al., 1999; Condorelli

et al., 1996; Kelliher et al., 1996; Hawley et al., 1997), under-

Increased expression of this oncogene was not associated with

cytogenetic abnormalities affecting the LYL1 locus (19p13),

con-standing of the downstream transcriptional programs that

gen-erate and maintain the T-ALL phenotype remains limited. Further

sistent with the paucity of reports on LYL1 activation by

chromo-somal translocation. Thus, other mechanisms appear

responsi-improvement of risk-based treatment strategies and the

devel-opment of effective new drugs for T-ALL will depend on fresh

ble for the aberrant expression of LYL1 in thymic leukemias.

Finally, TAL2 and BHLHB1 were expressed at high levels in a

insights into the molecular pathways usurped by HOX11, TAL1,

and other oncoproteins in developing thymocytes.

single case each (Figure 1C). Again, cytogenetic analysis failed

to reveal locus-specific translocations associated with the

ex-pression of these oncogenes. Some cases overexpressed more

Results

than one of the closely related bHLH T cell oncogenes: six

overexpressed both TAL1 and LYL1, and one overexpressed

Oncogenic transcription factor expression in T-ALL

HOX11, an orphan homeobox gene essential for splenic devel-

TAL1 and BHLHB1.

Analysis of the LIM-only domain genes LMO1 and LMO2

opment (Roberts et al., 1994; Dear et al., 1995), is activated

in a subset of T-ALL cases bearing the t(10;14)(q24;q11) or

(Figure 1D) showed an absence of significant expression of

either LMO gene in the HOX11

samples. However,

overexpres-t(7;10)(q35;q24), each of which places HOX11 under the control

of strong enhancers embedded in the T cell receptor loci. Acting

sion of one of these genes was observed in most samples

overexpressing TAL1, and high levels of LMO2, but not LMO1,

on the hypothesis that HOX11 might be aberrantly expressed

in cases other than those harboring locus-specific transloca-

were found in the LYL1

samples. These results are consistent

with biochemical and transgenic animal model studies showing

tions, we used quantitative real-time reverse transcriptase PCR

(RT-PCR) to analyze HOX11 expression, detecting high levels

that LMO proteins form heterocomplexes and act in concert

with TAL1 and possibly other bHLH proteins in T-ALL

(Valge-of this oncogene in 8 (Valge-of 59 pediatric T-ALL samples (Figure 1A).

Four of the HOX11-positive cases had cytogenetic abnor-

Archer et al., 1994; Wadman et al., 1994; Larson et al., 1996;

Chervinsky et al., 1999; Herblot et al., 2000; Wadman et al.,

malities involving the HOX11 locus in chromosome band

10q24: two t(10;14)(q24;q11.2), one t(7;10)(q35;q24), and one

1997). Ten of the 59 cases (samples 48–51, 53–57, and 59,

Figure 1) did not express abnormal levels of any of the

transcrip-del(10)(q24q26). Reduced levels of HOX11 expression (100–

1000 times lower than in the eight HOX11

samples) were de-

tion factor genes described above, raising the possibility of

thymocyte transformation via alternative oncogenic

mecha-tected in four additional samples, two of which also had

cytoge-netic abnormalities of band 10q24 [t(10;14) and add(10)(q24)].

nisms.

The remaining cases and eight normal control thymus samples

showed only background levels of HOX11 expression, near the

Gene expression profiles and their biologic correlates

T cell development is a tightly regulated multistep process that

limit of detection with this technique. Thus, by using quantitative

RT-PCR analysis of the HOX11 gene, we identified a substantial

involves the intrathymic differentiation, proliferation, and

selec-tion of T cell precursors (Murre, 2000; Rodewald and Fehling,

proportion of HOX11

cases that express high levels of the

oncogene while lacking cytogenetically detectable alterations

1998). Leukemic thymocytes retain many of the biologic features

of normal T cell precursor subpopulations, as illustrated by

of the 10q24 region.

Prominent among T cell oncoproteins are members of the

shared patterns of cell surface protein expression (Reinherz and

Schlossman, 1980; Bene et al., 1995). We therefore postulated

basic helix-loop-helix (bHLH) family of transcription factors:

TAL1, TAL2, LYL1, and the recently described BHLHB1 protein

that HOX11, TAL1, and LYL1 overexpression might directly or

indirectly interfere with transcriptional networks that normally

(Bernard et al., 1990; Finger et al., 1989; Xia et al., 1991;

Mellen-tin et al., 1989; Wang et al., 2000). These transcriptional regula-

regulate thymocyte proliferation, differentiation, and survival

during T cell development (Look, 1997). To test this hypothesis,

tors are believed to act through a common mechanism involving

dominant negative interference with the activities of the E47

we used oligonucleotide microarrays (Affymetrix, HU6800) to

analyze the global patterns of gene expression in 39 of the

and E12 variants of E2A transcription factors (Begley and Green,

1999; Wang et al., 2000; Miyamoto et al., 1996; Hsu et al., 1994;

T-ALL samples with sufficient RNA for these studies (Golub et

al., 1999). The microarray data are available in their entirety as

Park and Sun, 1998), whose homozygous inactivation leads

to T cell tumors in mice (Bain et al., 1997; Yan et al., 1997).

Supplemental Data at http://www.genome.wi.mit.edu/mpr and

http://www.cancercell.org/cgi/content/full/1/1/75/DC1.

Quantitative RT-PCR analysis revealed increased levels of TAL1

mRNA in 29 (49%) of the 59 cases (Figure 1B). None of these

We first asked whether the results obtained from microarray

hybridizations agreed with the quantitative RT-PCR findings

samples harbored any of the recurrent translocations of

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chro-Figure 1. Quantitative RT-PCR analysis of oncogenic transcription factor genes in pediatric T-ALL samples and normal thymus controls

A: HOX11 expression. Samples with expression levels⬎1 ⫻ 105mRNA copies/100 ng of RNA (dotted line) were considered HOX11. Cases with abnormalities of chromosome band 10q24 are indicated with arrows.

B: TAL1 expression. Crosshatched bars indicate samples with the Tal1d variant band in 1p32, resulting from deletion of a 90 kb genomic DNA fragment adjacent to the TAL1 locus. Samples showing TAL1 expression levels above that detected in TAL1dsamples were considered TAL1⫹(dotted line). C: Expression of LYL1 (yellow bars) and other bHLH transcription factor genes (TAL2 and BHLHB1) (green bars). The threshold level for LYL1 positivity (dotted line) corresponds to five times the mean level of expression in normal thymus control samples.

D: Expression of LMO1 (purple bars) and LMO2 (orange bars). The threshold level for LMO1 and LMO2 positivity (dotted line) corresponds to five times the mean level of LMO2 expression in normal thymus control samples. Oval symbols in panels A, B, and C indicate cases included in the microarray analysis.

presented in Figure 1. Normalized microarray results, plotted

sion of the CD1 (A-E family members), LAR, and CD10 genes

in a pattern resembling that of normal cells undergoing the early

as increasing intensities of red (positive) or blue (negative)

rela-tive to the mean value, are shown in Figure 2. The first row of

cortical stage of thymocyte differentiation (Terstappen et al.,

1992; Terszowski et al., 2001; Rodewald and Fehling, 1998).

colored squares in each of the three panels depicts the

expres-sion levels of HOX11 (top), TAL1 (middle), or LYL1 (bottom)

Many of the genes associated with HOX11 expression are

in-volved in cell growth and proliferation. These include adenosine

among the 27 cases independently expressing one of the three

major oncogenes. The cases are arranged in the same order

deaminase (target of pentostatin, fludarabine, and

2-chloro-deoxyadenosine), DNA topoisomerase (target of the

anthracy-used to display the quantitative RT-PCR data. There was

re-markable overall agreement between gene expression values

clines and epipodophyllotoxins), dihydrofolate reductase (target

of methotrexate), hypoxanthine phosphoribosyltransferase 1

obtained by these two methods.

We next surveyed genes considered to be “nearest neigh-

(modifier of the effect of antimetabolite therapy), and thymidylate

synthetase (target of fluoropyrimidines and other novel

folate-bors” (Golub et al., 1999) of HOX11, TAL1, and LYL1, based

on the close agreement of their expression profiles. Analysis of

based inhibitors). The gene products DNA polymerase epsilon,

cyclin A, Tax1 binding protein, and replication protein A1 all

the resultant gene expression signatures (Figure 2) revealed a

striking concordance with recognized stages of normal thymo-

have prominent roles in cell proliferation. These findings are

consistent with data showing that HOX11 can both immortalize

cyte development (Figure 3). Similar findings have been reported

for B lineage tumors studied with cDNA microarray technology

hematopoietic progenitors (Keller et al., 1998) and interact

di-rectly with cell cycle regulatory proteins (Kawabe et al., 1997).

(Allzadeh et al., 2000). HOX11

cases showed increased

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expres-For example, high levels of HOX11 correlated with increased

expression of MYC and the proapoptotic glucocorticoid

recep-tor gene. TAL1 overexpression was associated with the

upregu-lation of proto-oncogenes such as CBFA2 (AML1) and the

MYB-related gene MYBL2, receptor genes such as IL8R and CSFR1,

and the antiapoptotic gene BCL2A1. Finally, LYL1 positivity was

related to higher expression levels of the MYCN, LMO2, and

PLZF proto-oncogenes, as well as the antiapoptotic gene BCL2.

Most antineoplastic drugs are thought to act through the

mito-chondrial apoptotic machinery, and their cytostatic effects are

inhibited by BCL2 and its related prosurvival family members

(Reed, 1995). Thus, the upregulation of BCL2 and BCL2A1 in

LYL1- and TAL1-overexpressing cases may explain their relative

resistance to chemotherapy (see Figure 5), while the exquisite

Figure 3. Correlation of gene expression profiles in LYL1, HOX11, and TAL1

responsiveness of HOX11

cases could partly reflect the

down-T-ALL samples with recognized stages of thymocyte differentiation

regulation of survival factors in early cortical stage thymocytes,

Cell surface markers normally associated with each developmental stage

most of which are targeted for “death by neglect.”

(Murre, 2000) are indicated in black, with corresponding microarray findings shown in red. The most immature T cell precursors express CD34 but not

CD4, CD8, or CD3. As these cells mature, they lose CD34 expression while

Hierarchical clustering of T-ALL cases based on gene

gaining CD4 and then CD8, becoming double-positive thymocytes. Early

expression patterns

double-positive cells initially express CD1 and CD10 (early cortical

thymo-Although helping to identify the sets of genes coordinately

ex-cytes). As they finish rearranging their T cell receptor genes, 95% of these

cells fail to express a functional receptor and are ablated through a death-

pressed with HOX11, TAL1, and LYL1, the nearest neighbor

by-neglect mechanism. Thymocytes with functional T cell receptors gain

analysis depicted in Figure 2 provided little useful information

CD3 expression (late cortical thymocytes) and undergo both positive and

about the 10 cases that lacked discernible expression of these

negative selection. Cells surviving this process proceed through a final step

oncogenes (designated Other in Figure 4) or the two additional

of differentiation in which they downregulate the expression of either CD4

or CD8 to become mature single-positive T cells.

cases expressing both LYL1 and TAL1 (Mixed). To gain insight

in the molecular characteristics of these poorly understood

cases, we generated hierarchical clusters based on the 72 genes

whose expression patterns best distinguished between each

Because the drugs used to treat human leukemias are more

group of HOX11

, TAL1

, LYL1

, and other cases in pairwise

active in proliferating cells, these findings may explain in part

comparisons (as defined by the permutation distribution of the

the better prognosis of patients with HOX11

T-ALL (see Fig-

maximum t statistic, P

⬍ 0.30).

ure 5).

As shown in Figure 4, the HOX11

, TAL1

, and LYL

sam-By contrast, the expression pattern associated with TAL1

ples detected by RT-PCR are grouped together within major

expression appeared to reflect the late cortical stage of thymo-

branches of the dendrogram. The branch containing the HOX11

cyte differentiation, as indicated by the upregulation of LCK,

samples (H) comprises two subgroups, one containing most of

TCRA, TCRB, CD2, CD6, and CD3E (Terstappen et al., 1992;

the HOX11 RT-PCR-positive cases (H1) and the other consisting

Rodewald and Fehling, 1998). High levels of LYL1 expression

primarily of HOX11-like samples that lacked HOX11 expression

were associated with an undifferentiated thymocyte phenotype

by RT-PCR (H2). Surface immunophenotyping indicated that

characterized by increased expression of the early hematopoi-

these subgroups had related but distinct immunophenotypes

etic marker gene CD34, the cell adhesion gene L-selectin (SELL),

(Table 1). True HOX11 samples were primarily CD1

, CD10

⫹/⫺

,

the antiapoptotic gene BCL2, and LSP1, which encodes the

CD4

, CD8

, and CD3

(early cortical thymocytes), while the

lymphocyte-specific protein 1 (Pilarski et al., 1991; Galy et al.,

HOX11-like samples were primarily CD1

⫹/⫺

, CD10

, CD4

,

1993; Ma et al., 1995; Palker et al., 1998). These results suggest

CD8

, and CD3

(early cortical thymocytes with acquired CD3

that T cell oncogenes specifically interfere with transcriptional

surface expression). Similarly, the central cluster of TAL1

sam-programs controlling thymocyte development, leading to stage-

ples contained two subgroups, a larger one comprising most

specific developmental arrest.

of the true TAL1

samples (T1) and a second consisting mainly

The three molecularly distinct subtypes of T-ALL also

of TAL1-like samples (T2). A third, smaller branch (M) emerged

showed specific associations with known proto-oncogenes, as

from the hierarchical analysis, and was characterized by a global

pattern of increased expression of many of the genes that

distin-well as genes involved in programmed cell death (Figure 2).

Figure 2. HOX11, TAL1, and LYL1⫹nearest neighbor analysis

Each row of squares shows the expression pattern of a particular gene selected by nearest neighbor analysis (Golub et al., 1999), while each column represents 1 of the 27 samples positive for HOX11, TAL1, or LYL1 by RT-PCR (see Figure 1). The genes depicted were chosen from the top 200 nearest neighbors of each major oncogene (boldface type) on the basis of their potential functional relevance and then were grouped according to their involvement in T cell differentiation, apoptosis, cell proliferation, or chemotherapy response. Expression levels for each gene were normalized across the samples; levels greater than or less than the mean (by as much as three standard deviations) are shown in shades of red or blue, respectively. Numbers at the bottom correspond to the numbers of the samples in Figure 1. For a complete list of gene names, accession numbers, and raw expression values, see Supplemental Data at http://www.genome.wi.mit.edu/mpr and http://www.cancercell.org/cgi/content/full/1/1/75/DC1.

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Figure 4. Hierarchical cluster analysis of gene expression data

Expression profiles of 72 genes were selected by permutation test analysis as those best distinguishing among 39 HOX11, TAL1, LYL1⫹cases and unclassified samples (Other). The dendrogram (top) shows the relatedness of gene expression among samples and is color coded according to the quantitative RT-PCR category of each sample (see Figure 1). Clinical outcome data are reported as horizontal bars with open boxes representing survivors and dark boxes deceased patients. Cytogenetic and molecular abnormalities are indicated by discrete symbols defined at the bottom of the figure. Each column represents a T-ALL mRNA sample and each row a gene on the microarray. Genes are grouped into four consecutive categories: higher in HOX11than in TAL1⫹, LYL1, or Others; higher in TAL1than in HOX11, LYL1, or Others; higher in LYL1than in HOX11, TAL1⫹, or Others; and finally higher in Others than in HOX11, TAL1, or LYL1⫹; and are listed within each category in order from lowest to highest P value. A complete list of genes and P values is available as Supplemental Data at http://www.genome.wi.mit.edu/mpr and http://www.cancercell.org/cgi/content/full/1/1/75/DC1. Gene expression values are normalized and color coded, as indicated by the scale beneath the graph. Major branches in the dendrogram are designated by the first letter of the dominant oncogene (e.g., H, H1, H2, for HOX11).

guished among the other three groups. Interestingly, two of

Finally, the LYL

cluster (L) included two branches. One

contained three of the true LYL

samples (L1), including the

these three cases had the t(11;19)(q23;p13.3), which produces

the MLL-ENL fusion gene (Rubnitz et al., 1999a), while the re-

only T cell sample in this series with the FLT-3 internal tandem

duplication, which is often identified in acute myeloid leukemias

maining case had a normal karyotype. MLL-ENL RT-PCR

analy-sis, performed in 59 samples, revealed the MLL-ENL fusion

(Nakao et al., 1996; Yokota et al., 1997). These leukemias also

expressed high levels of CD34 as well as myeloid markers,

transcript in only three cases, all in the M cluster, including the

case with a normal karyotype. This result illustrates the power of

consistent with differentiation arrest in the early stages of T cell

development, when T progenitor cells are migrating from the

DNA microarray analysis to group samples according to specific

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Table 1. Cell surface antigen expression among T-ALL samples DPa DNb MYc CD34⫹ CD10⫹ CD1⫹ CD4⫹CD8⫹ CD4⫺CD8⫺ CD3⫹ CD13⫹/33⫹ Cluster H 43 78 75 78 0 36 7 n⫽ 14 HOX1137 62 100 62 0 0 0 In cluster H n⫽ 8 HOX11⫺ 50 100 50 100 0 83 16 In cluster H n⫽ 6 Cluster T 71 27 46 73 13 66 0 n⫽ 14 Cluster T1 78 20 40 70 20 90 0 n⫽ 9 Cluster T2 60 40 60 80 0 20 0 n⫽ 5 Cluster L 100 33 0 0 83 33 100 n⫽ 6

The values are percentages of positive samples. aDouble-positive thymocytes.

bDouble-negative thymocytes. cMyeloid lineage.

a LYL

sample and two samples with simultaneous expression

group of leukemias. HOX11L2, on the other hand, was

ex-pressed at high levels (

⬎10

6

copies per 100 ng of RNA) in six

of TAL1 and LYL1 by quantitative RT-PCR. Indications of

multistep mutational pathways emerged when samples were

of the T-ALL samples (29, 48, 49, 51, 54, and 55, as numbered

in Figure 1), but was undetectable in normal thymus and the

analyzed by quantitative DNA PCR for deletions of

P16/INK4A-P14/ARF (Drexler, 1998; Okuda et al., 1995). In our series of

other T-ALL samples. Three of the six HOX11L2

cases (closed

triangles in Figure 4) had sufficient RNA for microarray analysis.

T-ALL cases, homozygous deletions of this gene were found

in most samples in clusters H and T (Figure 4), which included

Their location in the HOX11-related “H2 cluster” of the

hierarchi-cal dendrogram confirms our hypothesis that cases with gene

HOX11

and TAL1

cases as well as cases with similar overall

patterns of gene expression. Homozygous deletion of P16/

expression signatures resembling HOX11

cases might be

transformed through the effects of highly related oncogenes

INK4A-P14/ARF was not detected in 2 of the three MLL-ENL

cases in the M cluster, nor in any of the cases grouped into

operating through similar oncogenic pathways.

Statistical analysis to identify the genes that were

differen-cluster L, which comprise LYL1

cases as well as mixed cases

expressing both TAL1 and LYL1 (Figure 4). The exclusive pres-

tially expressed in HOX11

versus HOX11L2

cases revealed

increased expression (Permax P value

⬍ 0.30, see Experimental

ence of cytogenetic features such as the 5q- and 13q-deletions

within the L2 subgroup (Figure 4) further attests to the ability

Procedures) of HOX11 itself and eight additional genes in

HOX11

cases (Fuse binding protein 2 [FBP2; U69126],

of our hierarchical clustering approach to group samples with

common mechanisms of transformation and suggests that tu-

DXS9879E [ITBA2; X92896], H2AZ histone [H2AZ; M37583],

glycerladehyde-3-phosphate

dehydrogenase

[G3PD,

EC

mor suppressor genes in the 5q and 13q regions are inactivated

as part of a distinct oncogenic pathway that gives rise to these

1.2.1.12; X01677], SW1/SNF complex 155 kDa subunit

[BAF155; U66615], FYN binding protein [FYB; U93049],

protea-leukemias.

some subunit

␣ 1 [PSMA1; M64992] and tubulin ␤ 5 [V00599]).

Oncogene discovery through microarray expression

None of the genes on the microarray were expressed at

signifi-analysis: HOX11L2 is activated in T-ALL samples

cantly higher levels in HOX11L2

cases. Thus, despite the

The observation that T-ALL leukemia cases with MLL-ENL re-

marked similarities in gene expression profiles between HOX11

arrangement and recurrent cytogenetic abnormalities were

and HOX11L2

cases, HOX11

cases are distinguished by

in-grouped together in our hierarchical clustering analysis illus-

creased expression of genes involved in signal transduction

trates the ability of gene expression profiling to identify cases

and the chromatin-mediated control of gene expression (see

with common mechanisms of transformation. Results of this

Discussion).

analysis also suggested that cases without defined oncogene

activation that clustered with the HOX11, TAL1, or LYL1 samples

Oncogene activation and gene expression signatures

have prognostic relevance

likely harbor related but as yet unidentified oncogenes. To test

this hypothesis, we used quantitative RT-PCR to analyze the

To assess the prognostic significance of these findings, we first

analyzed the survival of 58 eligible patients from the 59 whose

expression of HOX11L1 and HOX11L2, two homeobox genes

that are not included in the Affymetrix 6800 microarray but are

leukemic cells were analyzed for HOX11, HOX11L2, TAL1, or

LYL1 expression by quantitative RT-PCR. Preliminary

compari-functionally and structurally related to HOX11. HOX11L1 was

expressed at comparable low levels in both normal thymus and

son of the Kaplan-Meier plots showed no significant difference

between the TAL1

and LYL1

groups, prompting us to

com-T-ALL samples, indicating that it was not overexpressed in this

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achieve complete remission, was noted only in patients with

overexpression of TAL1 or LYL1.

Similar results were obtained when the Kaplan-Meier

analy-sis focused on the major groups defined by hierarchical

cluster-ing of gene expression signatures (Figure 5B): probability of

survival at 5 years was 92%

⫾ 8% for the HOX11

cluster

versus 43%

⫾ 19% and 33% ⫾ 19% for the TAL1

and LYL1

clusters (P

⫽ 0.03). None of the three patients in the small cluster

containing the MLL-ENL cases have died. Although based on

a small number of samples, this result agrees with a previous

report suggesting that MLL-ENL translocations in T-ALL may

not carry the dire prognosis associated with related

transloca-tions in infants and older children with an early B lineage ALL

immunophenotype (Behm et al., 1996; Rubnitz et al., 1999b).

Discussion

The dramatically different clinical courses of T-ALL patients

treated with the same intensive multidrug regimens support the

interpretation that ALL arising in thymic lymphocytes comprises

several biologically distinct diseases. This variability likely

re-flects a molecular heterogeneity that has not been appreciated

from characterization of leukemic T cells by conventional

meth-ods, as recently demonstrated in a microarray study for diffuse

large B cell lymphoma (Allzadeh et al., 2000). Thus, gene

expres-sion analysis using oligonucleotide or cDNA microarrays offers

a novel tool for delineating molecular pathways that drive the

malignant transformation of developing thymocytes.

The results reported here identify previously unrecognized

molecular subtypes of T-ALL and link the activation of particular

oncogenes to defined stages of normal thymocyte

develop-ment. Highly favorable clinical outcomes were observed for

patients in the HOX11

cluster, whose cell samples showed a

pattern of gene expression resembling that of early cortical

thymocytes. The better therapeutic responsiveness of this

sub-group may be explained by several distinctive features of

HOX11

lymphoblasts, including the expression of genes

asso-ciated with increased cell proliferative activity and the lack of

expression of BCL2 and related antiapoptotic genes. Apoptosis

is a major regulatory mechanism during normal T cell

develop-Figure 5. Clinically important T-ALL subgroups identified by gene expression

ment, eliminating the more than 90% of normal cortical

thymo-profiling

cytes unable to express functional T cell receptors (Vacchio et

A: Kaplan-Meier plots of overall survival among patients with a HOX11⫹,

al., 1998). Thus, HOX11

lymphoblasts appear to be arrested

HOX11L2, bHLH⫹, or unclassified (Other) gene expression signature by

RT-at a stage of thymocyte development thRT-at is especially

respon-PCR analysis (TAL1, TAL2, bHLHB1, and LYL1⫹samples combined as

bHLH⫹).

sive to drug-induced programmed cell death.

B: Kaplan-Meier plots of overall survival for subgroups recognized by hierar-

Less favorable outcomes were observed in subgroups

de-chical clustering of DNA microarray data, which subdivided the samples

fined by gene expression profiles characteristic of TAL1

or

into four main clusters (H, T, M, and L; see Figure 4). Tick marks on the curves

LYL1

samples, which resemble late cortical and early pro-T

represent surviving patients.

thymocytes, respectively. Drug resistance in LYL

samples may

be explained by the fact that early double-negative pro-T cells

express high levels of BCL2 and show increased resistance to

apoptosis (Veis et al., 1993). TAL1

cells appear to upregulate

bine these two cohorts (bHLH

) for further analysis. As shown

BCL2A1 (also known as BFL1) and other antiapoptotic

mole-in Figure 5A, constitutive expression of HOX11 was associated

cules normally induced by signaling through the TCR in late

with a favorable prognosis: probability of survival at 5 years was

cortical thymocytes (Tomayko et al., 1999), suggesting different

100%

⫾ 0% standard error (SE), compared with 30% ⫾ 24%

mechanisms of treatment resistance in TAL1

and LYL1

cases.

for the HOX11L2 group and 51%

⫾ 9% for the bHLH

group

We find it surprising that LYL1 and TAL1 overexpression is

(P

⫽ 0.02 by log-rank analysis, comparing HOX11

with all other

associated with maturational arrest at opposite ends of the

patients). Patients whose leukemic cells lacked expression of

thymocyte developmental spectrum, despite structural and

bio-HOX11, HOX11L2, or bHLH oncogenes had essentially the

chemical data suggesting that these two proteins might act

same probability of 5 year survival as the latter two groups (P

(9)

(Murre, 2000; Baer, 1993; Miyamoto et al., 1996). This may

would stress, however, that only a small subset of the genes

comprising each gene expression signature are likely to be

di-reflect differences in the stages of thymocyte differentiation

at which these oncogenes are activated. Alternatively, the key

rectly regulated by the oncogenic transcription factors

them-selves. Since many of the specifically expressed genes appear

transformation event involving bHLH oncogenes may occur

early in the CD4

/CD8

cell stage. In this model, TAL1 may

to reflect a specific stage of T cell developmental arrest, it will

be important to compare gene expression profiles in leukemic

abrogate the normal E2A-induced arrest of further differentiation

(Engel et al., 2001) more effectively than LYL1, leading to leuke-

T lymphoblasts versus subsets of normal thymocytes at different

stages of differentiation to identify transcriptional programs that

mias that resemble more mature CD4

/CD8

double-positive

thymocytes.

are directly linked to leukemic transformation.

Our studies indicate that wider application of gene

expres-Our microarray studies of leukemic thymocytes revealed

distinctive gene expression signatures that are strongly associ-

sion profiling in T-All would help to identify therapeutically

rele-vant diagnostic subgroups. It may also be possible, given

suffi-ated with specific oncogenic transcription factors. In some

in-stances, closely related signatures were found in samples lack-

cient numbers of patients, to identify signal transduction

pathways that are vital to the proliferation and survival of

individ-ing activation of known T-ALL oncogenes, leadindivid-ing us to predict

alternative oncogenic transcription factors that could initiate

ual subgroups, making proteins within these pathways attractive

targets for new therapeutic approaches.

similar patterns of gene expression. In experiments based on

this hypothesis, we identified HOX11L2 overexpression as an

Experimental procedures

oncogenic event in HOX11-negative samples that exhibited the

gene expression signature associated with bona fide

HOX11-Patient material

expressing cases. HOX11L2 is an orphan homeobox factor very

Samples of cryopreserved lymphoblasts from 59 children and young adults

similar to HOX11, and has been shown to be essential for the

with T-ALL, treated in Total Therapy studies XI–XIII at St. Jude Children’s

normal development of the ventral medullary respiratory center,

Research Hospital (TN), were obtained with informed consent at the time of diagnosis, before any chemotherapy was given. The median age of the

in that its deficiency in mice leads to a respiratory failure

resem-patients was 9.3 years (range 0.5–18.8), the male to female ratio was 3.0,

bling congenital central hypoventilation syndrome in humans

and the leukocyte count at diagnosis was 2,300–917,000 per mm3(median,

(Shirasawa et al., 2000). The recent report by Bernard et al.

164,000). Mean lymphoblast percentage in the samples analyzed was 91%⫾

(2001) of a novel cryptic recurrent translocation t(5;14)(q35;q32)

10% SD. Six patients had CNS disease at presentation, and mediastinal

in T-ALL resulting in aberrant HOX11L2 expression reinforces

masses were present in 36. One case with less than one year of followup

the role of this homeobox factor as a T-ALL oncogene.

was excluded from survival analysis. Lymphoid cells were also obtained (with informed consent) from normal thymic tissue removed at the time of

Given the marked similarity in gene expression profiles

be-cardiac surgery.

tween HOX11

and HOX11L2

cases, it is surprising that these

two groups of patients have such different treatment outcomes,

DNA and RNA preparation

and additional patients will need to be studied to confirm this

RNA was prepared from cryopreserved lymphoblasts with RNAqueous

re-result. Of the eight genes that were more highly expressed by

agents (Ambion) according to the manufacturer’s instructions and

quanti-HOX11

leukemias, FBP2, BAF155, and FYB encode regulatory

tated spectrophotometrically. The quality of the purified RNA was assessed by visualization of 18S and 28S RNA bands under ultraviolet light after

proteins that might provide insight into the dissimilar clinical

electrophoresis through denaturing agarose gels and staining with ethidium

responses. The far upstream binding protein 2 (FBP2) regulates

bromide. Genomic DNA from each sample was extracted with a commercial

alternative mRNA splicing through binding to intronic splicing

kit (GENTRA) following the manufacturer’s instructions,

spectrophotometri-enhancer sequences (Min et al., 1997), while BRG1-associated

cally quantified, and stored at⫺20⬚C until analysis.

factor 155 (BAF155) is the human homolog of the yeast protein

SWI3, a component of the SWI/SNF complex that regulates

Primers and probes

gene expression through chromatin remodeling. Two compo-

Primers and probes were designed with the assistance of the computer program Primer Express (Perkin-Elmer Applied Biosystems) and with flanking

nents of the corresponding mammalian complex, BAF47/SNF5

intron-exon boundaries to prevent amplification from any residual genomic

(Roberts et al., 2000; Versteege et al., 1998) and BRG1 (Wong

DNA, while avoiding areas involved in the generation of alternative spliced

et al., 2000), are known to be tumor suppressors, and BAF155

mRNAs. In the case of TAL2, BHLHB1, HOX11L1, and HOX11L2, which

together with BRG1 has been shown to interact with cyclin E,

lacked suitable intron-exon boundaries for primer-probe design, the amount

with BRG1 specifically causing cell growth arrest (Shanahan et

of residual genomic DNA in each sample was determined by simultaneous

al., 1999). FYN binding protein (FYB) is an important positive

quantitation of these genes on RNA specimens in the presence and absence of reverse transcriptase. GAPDH FW: 5⬘-GAAGGTGAAGGTCGGAGT-3⬘,

regulator of T cell activation and couples TCR signals to integrin

GAPDH RV: 5⬘-GAAGATGGTGATGGGATTTC-3⬘, GAPDG Probe:

5⬘-VIC-activation and adhesion (Geng et al., 2001; Griffiths et al., 2001;

CAAGCTTCCCGTTCTCAGCC-TAMRA-3⬘, TAL1 FW: 5⬘-GAAGAGGAGA

Peterson et al., 2001).

CCTTCCCCCT-3⬘, TAL1 RV: 5⬘-GGTGAAGATACGCCGCACA-3⬘, TAL1

In contrast to the inclusive microarray analysis employed

Probe: 5⬘-FAM-TGAGATGGAGATTACTGATGGTCCCCA-TAMRA-3⬘, TAL2

by Allzadeh and coworkers to characterize subgroups of B cell

FW: 5⬘-GCCTGCAACAAACGGGAGT-3⬘, TAL2 RV: 5⬘-AGAGTTCTGTCCTC

lymphoma (Allzadeh et al., 2000), we chose to focus genes that

CAGGCCT-3⬘, TAL2 Probe:

5⬘-FAM-CTCTTCCCTCAAGGACCCCACCTGC-best distinguish among cases expressing known T-All onco-

TAMRA-3⬘, LYL1 FW: CCCACTTTGGCCCTGCA-3⬘, LYL1 RV: 5⬘-GGTCCTGCTGGCCCAATGT3⬘, LYL1 Probe: 5⬘-FAM-TACCACCCTCACC

genes. This approach was based on the hypothesis that

domi-CCTTCCTCAACAGTGTC-TAMRA-3⬘ BHLHB1 FW: 5⬘-GGCAGTGGCTT

nant oncogenic transcription factors in this disease, such as

CAAGTCGTC-3⬘, BHLHB1 RV: 5⬘-TCCGGCTCTGTCATTTGCTT-3⬘,

HOX11, TAL1, and LYL1, stand at the top of regulatory cascades

BHLHB1 Probe: 5⬘-FAM-TCGTCCAGCACCTCGTCGTCTACG-TAMRA-3⬘,

whose aberrant activation can lead to T cell neoplasia. Our

HOX11 FW: 5⬘-TGGATGGAGAGTAACCGCAGAT-3⬘, HOX11 RV:

5⬘-“hypothesis driven” approach to hierarchical clustering has en-

GGGCGTCCGGTTCTGATA-3⬘, HOX11 Probe: 5⬘-FAM-CACAAAGGACAG

abled us to integrate complex gene expression patterns into a

GTTCACAGGTCACCC-TAMRA-3⬘, HOX11L1 FW: 5⬘-GGATGCTGGGTC CACACAAC-3⬘, HOX11L1 RV: 5⬘-CAGGATCTGATCGATGCCGA-3⬘,

conceptual framework with biologic relevance to T cell ALL. We

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HOX11L1 Probe: 5⬘-FAM-TCCCACACCACGAGCCAATCAGC-TAMRA-3⬘, Acknowledgments

HOX11L2 FW: 5⬘-GCCCAAGCGTAAGAAGCCGC-3⬘, HOX11L2 RV: 5⬘-AGC

We thank Pablo Tamayo for help with the microarray analysis, Michael GCTTTTCCAGCTCGCAG-3⬘, HOX11L2 Probe: 5⬘-FAM-CACGTCCTTTTC

Hancock and Yinmei Zhou for help with the prognostic analysis, David CCGGGTGCAGA-TAMRA-3⬘, LMO1 FW: 5⬘-TCTACACCAAGGCCAACC

Zahrieh for assistance with the hierarchical dendrograms, John Gilbert for TCA-3⬘, LMO1 Probe:

5⬘-FAM-CGCGACTACCTGAGGCTCTTTGGCA-editorial review and substantive comments, and Craig Bassing for critical TAMRA-3⬘, LMO1 RV:TGCAAGCAGCACAGTTCCC-3⬘, LMO2 FW:

5⬘-review of the manuscript. This work was supported by NIH grants CA 68484 TACAAACTGGGCCGGAAGC-3⬘, LMO2 Probe: 5⬘-FAM-CGGAGAGACTAT

and CA 21765, the American Lebanese Syrian Associated Charities (ALSAC), CTCAGGCTTTTTGGGC-TAMRA-3⬘, LMO2 RV:5⬘-CTTGTCACAGGATGCG

St. Jude Children’s Research Hospital, Bristol-Myers Squibb, Millennium CAGA-3⬘. Unmodified primers and 5⬘-FAM, 3⬘-TAMRA or 5⬘-HEX, 3⬘-TAMRA

Pharmaceuticals, and Affymetrix, Inc. A.A.F. is a Fellow of the Leukemia and labeled probes were synthesized by Integrated DNA Technologies, while

Lymphoma Society. GAPDH 5⬘-VIC, 3⬘-TAMRA labeled probe was synthesized by PE Applied

Biosystems. Tal1d, FLT3 ITD analysis by PCR and MLL-ENL RT-PCR fusion transcript detection were performed as previously described (Meshinchi et al., 2001; Pongers-Willemse et al., 1999; Rubnitz et al., 1996).

Received: December 28, 2001 Revised: January 17, 2002

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